改良联合酶消化法制备人瘢痕疙瘩组织单细胞悬液的实验研究

Preparation of Single-Cell Suspensions from Human Keloid Tissue by Modified Combined Enzyme Digestion

目的探讨改良的联合酶消化法对于瘢痕疙瘩真皮组织的消化效果,并探究最佳消化时间。方法收集瘢痕疙瘩样本8例,去除表皮后用剪刀将真皮组织匀浆。真皮匀浆采用A、B两种方案进行消化。A方案采用传统的联合酶消化法(Ⅰ型胶原酶+Ⅱ型胶原酶+Ⅳ型胶原酶+透明质酸酶);B方案采用改良联合酶消化法,在A方案的基础上添加了DNaseⅠ。采用显微镜计数单细胞占比和单细胞数,台盼蓝染色和流式细胞检测细胞活性,比较A、B两种消化方案的消化效率,同时探究B方案的最佳消化时间。结果消化2 h和4 h后,B方案相比A方案获得的单细胞比例显著升高(P<0.05),单细胞数显著增多(P<0.01)。应用B方案,消化时间延长至6 h,相比消化4 h得到的单细胞比例和单细胞数显著增加(P<0.05),细胞活性无明显改变(P>0.05)。消化时间延长至8 h和10 h,与6 h相比,单细胞比例和单细胞数无明显增加(P>0.05),细胞活性显著下降(P<0.05)。结论在传统联合酶消化液中添加DNaseⅠ能够提高消化效率,有效制备疤痕疙瘩组织的单细胞悬液。组织消化6 h能够在保持细胞活性的同时达到较好的消化效果。

Objective To investigate the effect of modified method of combined enzyme digestion for keloid dermis digestion and to explore the optimal digestion time.Methods Eight keloid tissue samples were collected.After the removal of the epidermis,dermis was homogenized with the scissor.The homogenates of the dermis were digested with group A or B protocol simultaneously.The group A formula was composed of traditional combined enzyme(typeⅠcollagenase+typeⅡcollagenase+typeⅣcollagenase+hyaluronidase),while the group B formula was supplemented with DNaseⅠ.Single-cell fraction and single-cell numbers were determined under the microscope.Cell viability was detected using Trypan blue staining and flow cytometry.The digestive efficiency between group A and B was compared.Meanwhile,the optimal digestion time for group B was investigated.Results After 2 hours and 4 hours digestion,the fraction(P<0.05)and number(P<0.01)of single cells obtained in group B were both significantly higher than that of group A.Compared with 4 hours digestion,the single-cell fraction and single-cell number were significantly increased when the digestion time was extended to 6 hours(P<0.05),whereas the cell viability was not significantly changed(P>0.05).Compared with 6 hours digestion,there was no significant increase in the single-cell fraction and single-cell number when the digestion time was extended to 8 hours or 10 hours(P>0.05),whereas the cell viability was decreased significantly(P<0.05).Conclusion The supplement of DNaseⅠto the traditional combined enzyme digestion can increase the digestive efficiency and thus,facilitate the preparation of single-cell suspension for keloid tissue.Tissue digestion for 6 hours can achieve satisfactory digestive results while maintaining the cell viability.